Intraventricular haemorrhage in premature infants: the role of immature neuronal salt and water transport.

Intraventricular hemorrhage (IVH) is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability, and/or hydrocephalus. Animal models suggest that brain tissue shrinkage with subsequent vascular stretch and tear is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for IVH are biomarkers of hypoxic-ischemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction under these conditions. This is because immature neurons express the chloride salt and water transporter NKCC1, which subserves regulatory volume increases in nonneural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic ischemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters will be diminished. As a consequence, while mature neurons swell (cytotoxic edema) immature neurons might shrink. After hypoxic-ischemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage, and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, as well as prevention of neuronal shrinkage and the ensuing IVH, in premature infants.

A Dynamic Balance between Neuronal Death and Clearance in an in Vitro Model of Acute Brain Injury.

In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.SIGNIFICANCE STATEMENT In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus, longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically expressed proteins, provide more accurate estimates of cell death than do single-time point anatomic or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

A dynamic balance between neuronal death and clearance after acute brain injury.

After acute brain injury, neuronal apoptosis may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. The size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for two weeks. Altering phagocytosis rates, e.g. by changing the number of microglia, dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for vital stains. Canonically neuroprotective interventions such as seizure blockade and neurotoxic maneuvers such as perinatal ethanol exposure were mediated by effects on microglial activity and the membrane permeability of apoptotic neurons, and had either no or opposing effects on healthy surviving neurons. Significance: After acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus longitudinal assays of healthy cells, such as assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays. More accurate estimates of death rates will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

GABAergic synaptic transmission regulates calcium influx during spike-timing dependent plasticity.

Coincident pre- and postsynaptic activity of hippocampal neurons alters the strength of gamma-aminobutyric acid (GABA(A))-mediated inhibition through a Ca(2+)-dependent regulation of cation-chloride cotransporters. This long-term synaptic modulation is termed GABAergic spike-timing dependent plasticity (STDP). In the present study, we examined whether the properties of the GABAergic synapses themselves modulate the required postsynaptic Ca(2+) influx during GABAergic STDP induction. To do this we first identified GABAergic synapses between cultured hippocampal neurons based on their relatively long decay time constants and their reversal potentials which lay close to the resting membrane potential. GABAergic STDP was then induced by coincidentally (±1 ms) firing the pre- and postsynaptic neurons at 5 Hz for 30 s, while postsynaptic Ca(2+) was imaged with the Ca(2+)-sensitive fluorescent dye Fluo4-AM. In all cases, the induction of GABAergic STDP increased postsynaptic Ca(2+) above resting levels. We further found that the magnitude of this increase correlated with the amplitude and polarity of the GABAergic postsynaptic current (GPSC); hyperpolarizing GPSCs reduced the Ca(2+) influx in comparison to both depolarizing GPSCs, and postsynaptic neurons spiked alone. This relationship was influenced by both the driving force for Cl(-) and GABA(A) conductance (which had positive correlations with the Ca(2+) influx). The spike-timing order during STDP induction did not influence the correlation between GPSC amplitude and Ca(2+) influx, which is likely accounted for by the symmetrical GABAergic STDP window.

Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize E(Cl) during development.

In the mature CNS, coincident pre- and postsynaptic activity decreases the strength of gamma-aminobutyric acid (GABA)(A)-mediated inhibition through a Ca2+-dependent decrease in the activity of the neuron-specific K+-Cl- cotransporter KCC2. In the present study we examined whether coincident pre- and postsynaptic activity can also modulate immature GABAergic synapses, where the Na+-K+-2Cl- (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl-](i)). Dual perforated patch-clamp recordings were made from cultured hippocampal neurons prepared from embryonic Sprague-Dawley rats. These recordings were used to identify GABAergic synapses where the reversal potential for Cl- (ECl) was hyperpolarized with respect to the action potential threshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within +/- 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of ECl by 10.03 +/- 1.64 mV, increasing the strength of synaptic inhibition. Blocking the inward transport of Cl- by NKCC1 with bumetanide (10 microm) hyperpolarized ECl by 16.14 +/- 4.8 mV, and prevented this coincident activity-induced shift of ECl. The bumetanide-induced hyperpolarization of ECl occluded furosemide, a K+-Cl- cotransporter antagonist, from producing further shifts in ECl. Together, this indicates that brief coincident pre- and postsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as a mechanism underlying inhibitory synaptic plasticity.

Extracellular potassium regulates the chloride reversal potential in cultured hippocampal neurons.

Inhibitory GABAergic synaptic transmission in the mammalian hippocampus depends upon a hyperpolarized reversal potential for Cl(-) (ECl). To examine the regulation of ECl hyperpolarization we cultured hippocampal neurons for two weeks in either a low- or a high-concentration of KCl (2.6 or 18.7 mM, respectively). Neurons were then recorded from standard extracellular solution containing 3 mM K+, using the dual perforated patch clamp technique. Low-KCl cultured neurons fired spontaneous action potentials (APs; 0.33+/-0.11 Hz), while high-KCl cultured neurons were quiescent, resulting in a significant difference in AP activity (p=0.042). This high-KCl-induced decrease in activity was accompanied by depolarizations of both the AP threshold (p<0.001) and ECl (p<0.001), and a decrease in input resistance (IR, p<0.001), when compared with low-KCl cultured neurons. Blocking AP firing of low-KCl neurons during culturing with 1 muM tetrodotoxin did not alter ECl hyperpolarization, when compared with drug-free cultured low-KCl neurons (p=0.627); thus AP firing is not required for ECl hyperpolarization. Acute perfusion of a high-KCl extracellular solution onto low- or high-KCl cultured neurons demonstrated that high-KCl significantly depolarized the resting membrane potential (RMP). The KCl-induced change in ECl did not correspond with alterations in the expression of the cation chloride cotransporters KCC2 and NKCC1, as determined by western blotting (p=0.736). These findings suggest that: (1) extracellular K+ regulates ECl hyperpolarization; and, (2) the use of high-KCl during neuronal culture produces biophysically abnormal parameters, and thus should be discouraged.